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It has been proposed that bile acid suppression of CYP7A1 gene expression is mediated through a gut-liver signaling pathway fibroblast growth factor (FGF)15/19-fibroblast growth factor receptor 4 which is initiated by activation of farnesoid X receptor in the ileum but not in the liver. This study evaluated whether FGF15/19 protein levels in the portal blood reflected changes in FGF15/19 mRNA in the ileum. Studies were conducted in Sprague Dawley rats and New Zealand white rabbits fed regular chow (controls), supplemented with cholesterol (Ch) or cholic acid (CA). After feeding CA, ileal FGF15 mRNA increased 8.5-fold in rats and FGF19 rose 16-fold in rabbits associated with 62 and 75% reduction of CYP7A1 mRNA, respectively. Neither FGF15 nor FGF19 protein levels changed in the portal blood to correspond with the marked increase of FGF15/19 mRNA levels in the ileum or inhibited CYP7A1 expression in the liver. Further, in Ch-fed rats, CYP7A1 mRNA increased 1.9-fold (P < 0.001) although FGF15 mRNA levels in the ileum and portal blood FGF15 protein levels were not decreased. In Ch-fed rabbits, although FGF19 mRNA levels in the ileum and liver did not increase significantly, CYP7A1 mRNA declined 49% (P < 0.05). We were unable to find corresponding changes of FGF15/19 protein levels in the portal blood in rats and rabbits where the mRNA levels of FGF15/19 in the ileum and CYP7A1 in the liver change significantly.

The transcription of the cholesterol 7α-hydroxylase gene (CYP7A1) is greatly decreased in cholesterol-fed rabbits. To determine whether the molecular structure of the promoter is responsible for this downregulation, we cloned the rabbit CYP7A1 promoter, identified the binding sites for α-fetoprotein transcription factor (FTF) and liver X receptor (LXRα), and studied the effects of FTF, LXRα, and SHP on its transcription. Adding LXRα/retinoid X receptor together with their ligands (L/R) to the promoter/reporter construct transfected into HepG2 cells greatly increased its activity. FTF did not increase promoter activity, nor did it enhance the stimulatory effect of L/R. Mutating the FTF binding site abolished the promoter baseline activity. Increasing amounts of SHP abolished the effect of L/R, and FTF enhanced the ability of SHP to decrease promoter activity below baseline levels. Thus, downregulation of CYP7A1 in cholesterol-fed rabbits is attributable secondarily to the activation of farnesoid X receptor, which increases SHP expression to override the positive effects of LXRα. Although FTF is a competent factor for maintaining baseline activity, it does not further enhance and may suppress CYP7A1 transcription.

Cerebrotendinous xanthomatosis (CTX), sterol 27-hydroxylase (CYP27A1) deficiency, is associated with markedly reduced chenodeoxycholic acid (CDCA), the most powerful activating ligand for farnesoid X receptor (FXR). We investigated the effects of reduced CDCA on FXR target genes in humans. Liver specimens from an untreated CTX patient and 10 control subjects were studied. In the patient, hepatic CDCA concentration was markedly reduced but the bile alcohol level exceeded CDCA levels in control subjects (73.5 vs. 37.8 ± 6.2 nmol/g liver). Cholesterol 7α-hydroxylase (CYP7A1) and Na+/taurocholate-cotransporting polypeptide (NTCP) were upregulated 84- and 8-fold, respectively. However, small heterodimer partner (SHP) and bile salt export pump were normally expressed. Marked CYP7A1 induction with normal SHP expression was not explained by the regulation of liver X receptor α (LXRα) or pregnane X receptor. However, another nuclear receptor, hepatocyte nuclear factor 4α (HNF4α), was induced 2.9-fold in CTX, which was associated with enhanced mRNA levels of HNF4α target genes, CYP7A1, 7α-hydroxy-4-cholesten-3-one 12α-hydroxylase, CYP27A1, and NTCP.In conclusion, the coordinate regulation of FXR target genes was lost in CTX. The mechanism of the disruption may be explained by a normally stimulated FXR pathway attributable to markedly increased bile alcohols with activation of HNF4α caused by reduced bile acids in CTX liver.

The CYP27A gene encodes a mitochondrial cytochrome P450 enzyme, sterol 27-hydroxylase, that is expressed in many different tissues and plays an important role in cholesterol and bile acid metabolism. In humans, CYP27A deficiency leads to cerebrotendinous xanthomatosis. To gain insight into the roles of CYP27A in the regulation of cholesterol and bile acid metabolism, cyp27A gene knockout heterozygous, homozygous, and wild-type littermate mice were studied. In contrast to homozygotes, heterozygotes had increased body weight and were mildly hypercholesterolemic, with increased numbers of lipoprotein particles in the low density lipoprotein size range. Cyp7A expression was not increased in heterozygotes but was in homozygotes, suggesting that parts of the homozygous phenotype are secondary to increased cyp7A expression and activity. Homozygotes exhibited pronounced hepatomegaly and dysregulation in hepatic cholesterol, bile acid, and fatty acid metabolism. Hepatic cholesterol synthesis and synthesis of bile acid intermediates were increased; however, side chain cleavage was impaired, leading to decreased bile salt concentrations in gallbladder bile. Expression of Na-taurocholate cotransporting polypeptide, the major sinusoidal bile salt transporter, was increased, and that of bile salt export pump, the major canalicular bile salt transporter, was decreased. Gender played a modifying role in the homozygous response to cyp27A deficiency, with females being generally more severely affected.Thus, both cyp27A genotype and gender affected the regulation of hepatic bile acid, cholesterol, and fatty acid metabolism.

We investigated how cholesterol feeding regulates cholesterol 7α-hydroxylase (CYP7A1) via the nuclear receptors farnesoid X receptor (FXR) and liver X receptor α (LXRα) in New Zealand white rabbits. After 1 day of 2% cholesterol feeding, when the bile acid pool size had not expanded, mRNA levels of the FXR target genes short-heterodimer partner (SHP) and sterol 12α-hydroxylase (CYP8B) were unchanged, indicating that FXR activation remained constant. In contrast, the mRNA levels of the LXRα target genes ATP binding cassette transporter A1 (ABCA1) and cholesteryl ester transfer protein (CETP) increased 5-fold and 2.3-fold, respectively, associated with significant increases in hepatic concentrations of oxysterols. Activity and mRNA levels of CYP7A1 increased 2.4 times and 2.2 times, respectively. After 10 days of cholesterol feeding, the bile acid pool size increased nearly 2-fold. SHP mRNA levels increased 4.1-fold while CYP8B declined 64%. ABCA1 mRNA rose 8-fold and CETP mRNA remained elevated. Activity and mRNA of CYP7A1 decreased 60% and 90%, respectively. Feeding cholesterol for 1 day did not enlarge the ligand pool size or change FXR activation, while LXRα was activated highly secondary to increased hepatic oxysterols. As a result, CYP7A1 was up-regulated. After 10 days of cholesterol feeding, the bile acid (FXR ligand) pool size increased, which activated FXR and inhibited CYP7A1 despite continued activation of LXRα.Thus, in rabbits, when FXR and LXRα are activated simultaneously, the inhibitory effect of FXR overrides the stimulatory effect of LXRα to suppress CYP7A1 mRNA expression.

Cholesterol 7α-hydroxylase, a rate-limiting enzyme for bile acid synthesis, has been implicated in genetic susceptibility to atherosclerosis. The gene, CYP7A1, encoding a protein with this activity, is expressed normally only in hepatocytes and is highly regulated. Our cyp7A1 gene knockout mouse colony, as young adults on a chow diet, is hypercholesterolemic. These mice were characterized extensively to understand how cyp7A1 affects lipid and bile acid homeostasis in different tissue compartments and whether gender plays a modifying role. Both male and female cyp7A1-deficient mice had decreased hepatic LDL receptors, unchanged hepatic cholesterol synthesis, increased intestinal cholesterol synthesis and bile acid transporters, and decreased fecal bile acids but increased fecal sterols. In females, cyp7A1 deficiency also caused changes in hepatic fatty acid metabolism, decreased hepatic canalicular bile acid transporter, Bsep, and gallbladder bile composition altered to a lithogenic profile.Taken together, the data suggest that cyp7A1 deficiency results in a proatherogenic phenotype in both genders and leads to a prolithogenic phenotype in females.

Mammalian physiological processes can distinguish between dietary cholesterol and non-cholesterol, retaining very little of the non-cholesterol in their bodies. We have recently identified two genes, ABCG5 and ABCG8, encoding sterolin-1 and -2 respectively, mutations of which cause the human disease sitosterolemia. We report here the mouse cDNAs and genomic organization of Abcg5 and Abcg8. Both genes are arranged in an unusual head-to-head configuration, and only 140 bases separate their two respective start-transcription sites. A single TATA motif was identified, with no canonical CCAT box present between the two genes. The genes are located on mouse chromosome 17 and this complex spans no more than 40 kb. Expression of both genes is confined to the liver and intestine. For both genes, two different sizes of transcripts were identified which differ in the lengths of their 3′ UTRs. Additionally, alternatively spliced forms for Abcg8 were identified, resulting from a CAG repeat at the intron 1 splice-acceptor site, causing a deletion of a glutamine. We screened 20 different mouse strains for polymorphic variants. Although a large number of polymorphic variants were identified, strains reported to show significant differences in cholesterol absorption rates did not show significant genomic variations in Abcg5 or Abcg8.—Lu, K., M-H. Lee, H. Yu, Y. Zhou, S. A. Sandell, G. Salen, and S. B. Patel. Molecular cloning, genomic organization, genetic variations, and characterization of murine sterolin genes Abcg5 and Abcg8. J. Lipid Res. 2002. 43: 565–578.

We investigated the role of the orphan nuclear receptor farnesoid X receptor (FXR) in the regulation of cholesterol 7α-hydroxylase (CYP7A1), using an in vivo rabbit model, in which the bile acid pool, which includes high affinity ligands for FXR, was eliminated. After 7 days of bile drainage, the enterohepatic bile acid pool, in both New Zealand White and Watanabe heritable hyperlipidemic rabbits, was depleted. CYP7A1 activity and mRNA levels increased while FXR was deactivated as indicated by reduced FXR protein and changes in the expression of target genes that served as surrogate markers of FXR activation in the liver and ileum, respectively. Hepatic bile salt export pump mRNA levels and ileal bile acid-binding protein decreased while sterol 12α-hydroxylase and sodium/taurocholate cotransporting polypeptide mRNA levels increased in the liver. In addition, hepatic FXR mRNA levels decreased significantly. The data, taken together, indicate that FXR was deactivated when the bile acid pool was depleted such that CYP7A1 was upregulated. Further, lack of the high affinity ligand supply was associated with downregulation of hepatic FXR mRNA levels. —Xu, G., L-x. Pan, S. K. Erickson, B. M. Forman, B. L. Shneider, M. Ananthanarayanan, X. Li, S. Shefer, N. Balasubramanian, L. Ma, H. Asaoka, S. R. Lear, L. B. Nguyen, I. Dussault, F. J. Suchy, G. S. Tint, and G. Salen. Removal of the bile acid pool upregulates cholesterol 7α-hydroxylase by deactivating FXR in rabbits. J. Lipid Res. 2002. 43: 45–50.

To study the effect of cholecystectomy on the regulation of classic and alternative bile acid syntheses, gallbladder-intact (n = 20) and cholecystectomized (n = 20) New Zealand White rabbits were fed either chow or chow with 2% cholesterol (3 g/day). After 10 days, bile fistulas were constructed in half of each rabbit group to recover and measure the bile acid pool and biliary bile acid flux. After cholesterol feeding, the bile acid pool size increased from 268 ± 55 to 444 ± 77 mg (P < 0.01) with a 2-fold rise in the biliary bile acid flux in intact rabbits but did not expand the bile acid pool (270 ± 77 vs. 276 ± 62 mg), nor did the biliary bile acid flux increase in cholecystectomized rabbits. Ileal apical sodium-dependent bile acid transporter protein increased 46% from 93 ± 6 to 136 ± 23 units/mg (P < 0.01) in the intact rabbits but did not change in cholecystectomized rabbits (104 ± 14 vs. 99 ± 19 units/mg) after cholesterol feeding. Cholesterol 7α-hydroxylase activity was inhibited 59% (P < 0.001) while cholesterol 27-hydroxylase activity rose 83% (P < 0.05) after cholesterol feeding in the intact rabbits but neither enzyme activity changed significantly in cholesterol-fed cholecystectomized rabbits. Fecal bile acid outputs reflecting bile acid synthesis increased significantly in the intact but not in the cholecystectomized rabbits fed cholesterol. Removal of the gallbladder prevented expansion of the bile acid pool after cholesterol feeding as seen in intact rabbits because ileal bile acid transport did not increase. As a result, cholesterol 7α-hydroxylase was not inhibited. —Xu, G., G. Salen, B. L. Shneider, M. Ananthanarayanan, S. Shefer, L. Ma, A. Batta, L. B. Nguyen, J. J. Lingutla, G. S. Tint, M. Pcolinsky, and F. J. Suchy. Cholecystectomy prevents expansion of the bile acid pool and inhibition of cholesterol 7α-hydroxylase in rabbits fed cholesterol.

We examined the effect of hyodeoxycholic acid (HDCA) on plasma cholesterol levels and atherosclerosis in mice. In wild-type C57BL/6 mice, feeding increasing amounts of HDCA resulted in i) progressive decrease in dietary cholesterol absorption, ii) increased concentrations of HDCA in the gallbladder bile, iii) decreased liver cholesterol content, iv) increased liver cholesterol synthesis, and v) increased plasma concentrations of HDCA. In C57BL/6 LDL-receptor knockouts (LDLR-KO) the addition of HDCA to chow and a 0.5% cholesterol diet decreased their total plasma cholesterol levels by 21% and 62%, respectively, because of a decrease in VLDL and LDL cholesterol. Turnover studies showed that HDCA has no effect on VLDL removal from plasma. Furthermore, the addition of HDCA to chow- and 0.5% cholesterol-fed LDLR-KO mice decreased the aortic root atherosclerosis lesion area by 50% and 80%, respectively. Finally, we tested the effect of HDCA on intestinal tumor formation. Feeding C57BL/6 ApcMin mice with HDCA did not affect the number of tumors but decreased the tumor volume in these animals. These results suggest that HDCA might have beneficial effects in the treatment of increased plasma cholesterol levels and atherosclerosis. —Sehayek, E., J. G. Ono, E. M. Duncan, A. K. Batta, G. Salen, S. Shefer, L. B. Neguyen, K. Yang, M. Lipkin, and J. L. Breslow. Hyodeoxycholic acid efficiently suppresses atherosclerosis formation and plasma cholesterol levels in mice.

The effects of feeding cholesterol, sitosterol, and lovastatin on cholesterol absorption, biosynthesis, esterification, and LDL receptor function were examined in the rat jejunal mucosa. Cholesterol absorption was measured by the dual-isotope plasma ratio method; the rate-limiting enzyme of cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, was measured as total and expressed enzyme activities (in the absence and presence of a phosphatase inhibitor, NaF, respectively); mucosal total and esterified cholesterol concentrations were determined by gas-liquid chromatography; LDL receptor function was assayed as receptor-mediated binding of 125I-labeled LDL to mucosal membranes. Feeding 2% sitosterol or 0.04% lovastatin for 1 week significantly (P < 0.01) decreased the amounts of cholesterol absorbed per day (−85% and −63%, respectively). In contrast, feeding 2% cholesterol for 1 week increased the amounts of absorbed cholesterol 27-fold, even though the percent absorption significantly decreased. With all three treatments, there was a coordinate regulation of total HMG-CoA reductase activity and receptor-mediated LDL binding. Cholesterol feeding downregulated both total jejunal HMG-CoA reductase activity (P < 0.05) and receptor-mediated LDL binding (P < 0.01), whereas lovastatin- and sitosterol-supplemented diets significantly upregulated both of these parameters. In the control, cholesterol-fed, and sitosterol-fed animals, about half of the total jejunal HMG-CoA reductase activity was expressed (in functional dephosphorylated form). However, in the lovastatin-treated rats with 4-fold stimulation of HMG-CoA reductase, only 23% of the total enzyme activity was expressed. Changes in total HMG-CoA reductase activity and receptor-mediated LDL binding in all tested groups occurred with no change in total concentrations of mucosal cholesterol, and only cholesterol-fed animals had increased mucosal esterified cholesterol concentrations. Thus, in response to various fluxes of dietary or newly formed cholesterol, HMG-CoA reductase and receptor-mediated LDL binding are coordinately regulated to maintain constant cellular cholesterol concentrations in the jejunum. —Nguyen, L. B., S. Shefer, G. Salen, G. S. Tint, F. Ruiz, and J. Bullock. Mechanisms for cholesterol homeostasis in rat jejunal mucosa: effects of cholesterol, sitosterol, and lovastatin.

Cerebrotendinous xanthomatosis (CTX) is a rare autosomal recessive disorder of bile acid biosynthesis. Clinically, CTX patients present with tendon xanthomas, juvenile cataracts, and progressive neurological dysfunction and can be diagnosed by the detection of elevated plasma cholestanol levels. CTX is caused by mutations affecting the sterol 27-hydroxylase gene (CYP27). CTX has been identified in a number of populations, but seems to have a higher prevalence in the Japanese, Sephardic Jewish, and Italian populations. We have assembled 12 previously unreported pedigrees from the United States. The CYP27 locus had been previously mapped to chromosome 2q33-qter. We performed linkage analyses and found no evidence of genetic heterogeneity. All CTX patients showed segregation with the CYP27 locus, and haplotype analysis and recombinant events allowed us to precisely map CYP27 to chromosome 2q35, between markers D2S1371 and D2S424. Twenty-three mutations were identified from 13 probands analyzed thus far; 11 were compound heterozygotes and 2 had homozygous mutations. Of these, five are novel mutations [Trp100Stop, Pro408Ser, Gln428Stop, a 10-base pair (bp) deletion in exon 1, and a 2-bp deletion in exon 6 of the CYP27 gene]. Three-dimensional structural modeling of sterol 27-hydroxylase showed that, while the majority of the missense mutations disrupt the heme-binding and adrenodoxin-binding domains critical for enzyme activity, two missense mutations (Arg94Trp/Gln and Lys226Arg) are clearly located outside these sites and may identify a potential substrate-binding or other protein contact site. —Lee, M-H., S. Hazard, J. D. Carpten, S. Yi, J. Cohen, G.T. Gerhardt, G. Salen, and S. B. Patel. Fine-mapping, mutation analyses, and structural mapping of cerebrotendinous xanthomatosis in U.S. pedigrees.

Cerebrotendinous xanthomatosis (CTX) is a rare, recessively inherited lipid storage disease characterized by a markedly reduced production of chenodeoxycholic acid and an increased formation of 25-hydroxylated bile alcohols and cholestanol. Patients with this disease are known to have mutations in the sterol 27-hydroxylase (Cyp27) gene. However, one study showed that mice with a disrupted Cyp27 gene did not have any CTX-related clinical or biochemical abnormalities. To explore the reason, hepatic cholesterol, cholestanol, and 12 intermediates in bile acid biosynthetic pathways were quantified in 10 Cyp27−/− and 7 Cyp27−/− mice, two CTX patients (untreated and treated with chenodeoxycholic acid), and four human control subjects by high resolution gas chromatography-mass spectrometry. Mitochondrial 27-hydroxycholesterol and 5β-cholestane-3α,7α,12α,27-tetrol were virtually absent in both Cyp27−/− mice and CTX patients. In Cyp27−/− mice, microsomal concentrations of intermediates in the early bile acid biosynthetic pathway (7α-hydroxycholesterol, 7α-hydroxy-4-cholesten-3-one, 7α,12α-dihydroxy-4-cholesten-3-one, and 5β-cholestane-3α,7α,12α-triol), 25-hydroxylated bile alcohols (5β-cholestane-3α,7α,12α,25-tetrol, 5β-cholestane-3α,7α,12α,23R,25-pentol, and 5β-cholestane-3α,7α12α,24R, 25-pentol), and cholestanol were all significantly elevated compared with those in Cyp27−/− mice, although the levels were lower than those in untreated CTX patients. The intermediate levels in early bile acid biosynthesis were more elevated in male (16–86% of CTX) than in female Cyp27−/− mice (7–30% of CTX). In contrast, 25-hydroxylated bile alcohol concentrations were not significantly different between male and female Cyp27−/− mice and were considerably lower (less than 14%) than those in CTX patients. These results suggest that 1) in Cyp27−/− mice, especially in females, classic bile acid biosynthesis via 7α-hydroxycholesterol is not stimulated as much as in CTX patients; and 2) formed 25-hydroxylated bile alcohols are more efficiently metabolized in Cyp27−/− mice than in CTX patients. —Honda, A., G. Salen, Y. Matsuzaki, A. K. Batta, G. Xu, E. Leitersdorf, G. S. Tint, S. K. Erickson, N. Tanaka, and S. Shefer. Differences in hepatic levels of intermediates in bile acid biosynthesis between Cyp27−/− mice and CTX.

We measured the percent absorption, turnover, and distribution of campestanol (24-methyl-5α-cholestan-3β-ol) in a sitosterolemic homozygote, her obligate heterozygous mother, and three healthy human control subjects. For reasons relating to sterol hyperabsorption, the homozygote consumed a diet low in plant sterols that contained campestanol at about 2 mg/day. The heterozygote and three control subjects were fed a diet supplemented with a spread that contained campestanol at 540 mg/day and sitostanol (24-ethyl-5α-cholestan-3β-ol) at 1.9 g/day as fatty acid esters. Plasma campestanol concentrations determined by capillary gasliquid chromatography were 0.72 ± 0.03 mg/dl in the homozygote, 0.09 ± 0.04 mg/dl in the heterozygote, and 0.05 ± 0.03 mg/dl for the control mean. After simultaneous pulse labeling with [3α-3H]campestanol intravenously and [23-14C]campestanol orally, the maximum percent absorption measured by the plasma dual-isotope ratio method as a single time point was 80% in the homozygote, 14.3% in the heterozygote, and 5.5 ± 4.3% as the mean for three control subjects. Turnover (pool size) values estimated by mathematical analysis of the specific activity versus time [3α-3H]campestanol decay curves were as follows: 261 mg in the homozygote, 27.3 mg in the heterozygote, and 12.8 ± 7.6 mg in the three control subjects (homogygote vs. controls, P < 0.001). The calculated production rate (mg/24 h) equivalent to actual absorption in the presence of dietary sterols and stanols was 0.67 mg/day or 31% of intake in the homozygote, 2.1 mg/day or 0.3% of intake in the heterozygote, and 0.7 ± 0.3 mg/day or 0.1% of intake in the three control subjects. However, the excretion constant from pool A (KA) was prolonged markedly in the homozygote, but was 100 times more rapid in the heterozygote and three control subjects. Thus, campestanol, like other noncholesterol sterols, is hyperabsorbed and retained in sitosterolemic homozygotes. However, campestanol absorption was only slightly increased in the sitosterolemic heterozygote and removal was as rapid as in control subjects.—Salen, G., G. Xu, G. S. Tint, A. K. Batta, and S. Shefer. Hyperabsorption and retention of campestanol in a sitosterolemic homozygote: comparison with her mother and three control subjects. J. Lipid Res. 2000. 41: 1883–1889.

Abstract: In classic cholic acid biosynthesis, a series of ring modifications of cholesterol precede side chain cleavage and yield 5β-cholestane-3α, 7α, 12α-triol. Side chain reactions of the triol then proceed either by the mitochondrial 27-hydroxylation pathway or by the microsomal 25-hydroxylation pathway. We have developed specific and precise assay methods to measure the activities of key enzymes in both pathways, 5β-cholestane-3α, 7α, 12α-triol 25- and 27-hydroxylases and 5β-cholestane-3α, 7α, 12α, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases. The extracts from either the mitochondrial or microsomal incubation mixtures were purified by means of a disposable silica cartridge column, derivatized into trimethylsilyl ethers, and quantified by gas chromatography–mass spectrometry with selected-ion monitoring in a high resolution mode. Compared with the addition of substrates in acetone, those in 2-hydroxypropyl-β-cyclodextrin increased mitochondrial triol 27-hydroxylase activity 132% but decreased activities of the enzymes in microsomal 25-hydroxylation pathway (triol 25-hydroxylase and 5β-cholestane-3α, 7α, 12α, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases) 13–60% in human liver. The enzyme activities in both pathways were generally 2- to 4-times higher in mouse and rabbit livers compared with human liver. In all species, microsomal triol 25-hydroxylase activities were 4- to 11-times larger than mitochondrial triol 27-hydroxylase activities but the activities of tetrol 24S-hydroxylase were similar to triol 27-hydroxylase activities in our assay conditions. The regulation of both pathways in rabbit liver was studied after bile acid synthesis was perturbed. Cholesterol feeding up-regulated enzyme activities involved in both 25- (64–142%) and 27- (77%) hydroxylation pathways, while bile drainage up-regulated only the enzymes in the 25-hydroxylation pathway (178–371%). Using these new assays, we demonstrated that the 25- and 27-hydroxylation pathways for cholic acid biosynthesis are more active in mouse and rabbit than human livers and are separately regulated in rabbit liver. —Honda, A., G. Salen, S. Shefer, Y. Matsuzaki, G. Xu, A. K. Batta, G. S. Tint, and N. Tanaka. Regulation of 25- and 27-hydroxylation side chain cleavage pathways for cholic acid biosynthesis in humans, rabbits, and mice: assay of enzyme activities by high-resolution gas chromatography–mass spectrometry. J. Lipid Res. 2000. 41: 442–451.

We investigated the effect of ileal bile acid transport on the regulation of classic and alternative bile acid synthesis in cholesterol-fed rats and rabbits. Bile acid pool sizes, fecal bile acid outputs (synthesis rates), and the activities of cholesterol 7α-hydroxylase (classic bile acid synthesis) and cholesterol 27-hydroxylase (alternative bile acid synthesis) were related to ileal bile acid transporter expression (ileal apical sodium-dependent bile acid transporter, ASBT). Plasma cholesterol levels rose 2.1-times in rats (98 ± 19 mg/dl) and 31-times (986 ± 188 mg/dl) in rabbits. The bile acid pool size remained constant (55 ± 17 mg vs. 61 ± 18 mg) in rats but doubled (254 ± 46 to 533 ± 53 mg) in rabbits. ASBT protein expression did not change in rats but rose 31% (P < 0.05) in rabbits. Fecal bile acid outputs that reflected bile acid synthesis increased 2- and 2.4-times (P < 0.05) in cholesterol-fed rats and rabbits, respectively. Cholesterol 7α-hydroxylase activity rose 33% (24 ± 2.4 vs. 18 ± 1.6 pmol/mg/min, P < 0.01) and mRNA levels increased 50% (P < 0.01) in rats but decreased 68% and 79%, respectively, in cholesterol-fed rabbits. Cholesterol 27-hydroxylase activity remained unchanged in rats but rose 62% (P < 0.05) in rabbits. Classic bile acid synthesis (cholesterol 7α-hydroxylase) was inhibited in rabbits because an enlarged bile acid pool developed from enhanced ileal bile acid transport. In contrast, in rats, cholesterol 7α-hydroxylase was stimulated but the bile acid pool did not enlarge because ASBT did not change. Therefore, although bile acid synthesis was increased via different pathways in rats and rabbits, enhanced ileal bile acid transport was critical for enlarging the bile acid pool size that exerted feedback regulation on cholesterol 7α-hydroxylase in rabbits. —Xu, G., B. L. Shneider, S. Shefer, L. B. Nguyen, A. K. Batta, G. S. Tint, M. Arrese, S. Thevananther, L. Ma, S. Stengelin, W. Kramer, D. Greenblatt, M. Pcolinsky, and G. Salen. Ileal bile acid transport regulates bile acid pool, synthesis, and plasma cholesterol levels differently in cholesterol-fed rats and rabbits.

The Smith-Lemli-Opitz syndrome (SLOS) is a congenital birth defect syndrome caused by a deficiency of 3β-hydroxysterol Δ7-reductase, the final enzyme in the cholesterol biosynthetic pathway. The patients have reduced plasma and tissue cholesterol concentrations with the accumulation of 7-dehydrocholesterol and 8-dehydrocholesterol. Bile acid synthesis is reduced and unnatural cholenoic and cholestenoic acids have been identified in some SLOS patients. To explore the mechanism of the abnormal bile acid production, the activities of key enzymes in classic and alternative bile acid biosynthetic pathways (microsomal cholesterol 7α-hydroxylase and mitochondrial sterol 27-hydroxylase) were measured in liver biopsy specimens from two mildly affected SLOS patients. The effects of 7- and 8-dehydrocholesterols on these two enzyme activities were studied by using liver from SLOS model rats that were treated with the Δ7-reductase inhibitor (BM15.766) for 4 months and were comparable with more severe SLOS phenotype in plasma and hepatic sterol compositions. In the SLOS patients, cholesterol 7α-hydroxylase and sterol 27-hydroxylase were not defective. In BM15.766-treated rats, both enzyme activities were lower than those in control rats and they were competitively inhibited by 7- and 8-dehydrocholesterols. Rat microsomal cholesterol 7α-hydroxylase did not transform 7-dehydrocholesterol or 8-dehydrocholesterol into 7α-hydroxylated sterols. In contrast, rat mitochondrial sterol 27-hydroxylase catalyzed 27-hydroxylation of 7- and 8-dehydrocholesterols, which were partially converted to 3β-hydroxycholestadienoic acids. Addition of microsomes to the mitochondrial 27-hydroxylase assay mixture reduced 27-hydroxydehydrocholesterol concentrations, which suggested that 27-hydroxydehydrocholesterols were further metabolized by microsomal enzymes. These results suggest that reduced normal bile acid production is characteristic of severe SLOS phenotype and is caused not only by depletion of hepatic cholesterol but also by competitive inhibition of cholesterol 7α-hydroxylase and sterol 27-hydroxylase activities by accumulated 7- and 8-dehydrocholesterols. Unnatural bile acids are synthesized mainly by the alternative pathway via mitochondrial sterol 27-hydroxylase in SLOS.— Honda, A., G. Salen, S. Shefer, A. K. Batta, M. Honda, G. Xu, G. S. Tint, Y. Matsuzaki, J. Shoda, and N. Tanaka. Bile acid synthesis in the Smith-Lemli-Opitz syndrome: effects of dehydrocholesterols on cholesterol 7α-hydroxylase and 27-hydroxylase activities in rat liver. J. Lipid Res. 1999. 40: 1520–1528.

A simple method for the gas–liquid chromatographic quantitation of human fecal bile acids and sterols is described where bile acids are subjected to n-butyl ester derivatization, without prior isolation from the stool, followed by trimethylsilylation of the sterols and bile acids. Under these conditions, bile acid derivatives are well resolved from each other and from the trimethylsilyl ether derivatives of fecal sterols and no overlap occurs. The method was shown to be highly reproducible and recoveries were similar to those obtained with other methods used for fecal bile acid analysis. Application of the method for bile acid and sterol analysis in human stool is described.—Batta, A. K., G. Salen, K. R. Rapole, M. Batta, P. Batta, D. Alberts, and D. Earnest. Highly simplified method for gas–liquid chromatographic quantitation of bile acids and sterols in human stool. J. Lipid Res. 1999. 40: 1148–1154.

5-Aminosalicylic acid conjugate of ursodeoxycholic acid was synthesized in above 90% yield by adding a basic solution of 5-aminosalicylic acid into the mixed anhydride formed with ursodeoxycholic acid and ethyl chloroformate. The 5-aminosalicylic acid conjugate of ursodeoxycholic acid was poorly secreted into the bile and was deconjugated with cholylglycine hydrolase and Clostridium perfringens, that deconjugate naturally occurring glycine and taurine conjugates of bile acids. However, ursodeoxycholic acid 5-aminosalicylic acid conjugate was not absorbed from the duodenum but was concentrated in the colon where it was partially hydrolyzed by the intestinal bacteria to ursodeoxycholic acid and 5-aminosalicylic acid. We believe that this unique conjugation of ursodeoxycholic acid with 5-aminosalicylic acid may facilitate the transport of both 5-aminosalicylic acid and ursodeoxycholic acid to the colon and may be useful for the treatment of colonic inflammatory bowel diseases, ulcerative colitis and Crohn's disease.—Batta, A. K., G. S. Tint, G. Xu, S. Shefer, and G. Salen. Synthesis and intestinal metabolism of ursodeoxycholic acid conjugate with an antiinflammatory agent, 5-aminosalicylic acid.

The effect of cholesterol feeding (3 g/day) on bile acid synthesis was examined in 10 New Zealand white rabbits (NZW), 8 Watanabe heterozygous and 10 homozygous rabbits with partial and complete deficiencies of LDL receptors. After 10 days of cholesterol feeding, bile fistulas were constructed and bile acid pool sizes were measured. Cholesterol feeding increased plasma and hepatic cholesterol levels in all rabbit groups. Baseline bile acid pool sizes were smaller (P < 0.01) in heterozygotes (139 ± 3 mg) and homozygotes (124 ± 30 mg) than NZW rabbits (254 ± 44 mg). After feeding cholesterol, bile acid pool sizes doubled with increased cholic acid synthesis in NZW and, to a lesser extent, in Watanabe heterozygous rabbits but not in homozygotes. Baseline cholesterol 7α-hydroxylase activity in NZW and heterozygotes declined 69% and 53% (P < 0.001), respectively, after cholesterol feeding. Sterol 27-hydroxylase activity reflecting alternative bile acid synthesis increased 66% (P < 0.01) in NZW and 37% in Watanabe heterozygotes but not in homozygotes after feeding cholesterol. Bile fistula drainage stimulated cholesterol 7α-hydroxylase activity but not sterol 27-hydroxylase activity in all thr ee rabbit groups. These results demonstrated that dietary cholesterol increased hepatic sterol 27-hydroxylase activity and alternative bile acid synthesis to expand the bile acid pool and inhibited cholesterol 7α-hydroxylase in NZW and in Watanabe heterozygous rabbits but not in homozygotes with absent hepatic LDL receptor function. Thus, in rabbits, sterol 27-hydroxylase is up-regulated by the increased hepatic cholesterol that enters the liver via LDL receptors whereas cholesterol 7α-hydroxylase is controlled by the circulating hepatic bile acid flux.—Xu, G., G. Salen, S. Shefer, G. S. Tint, L. B. Nguyen, T. T. Parker, T. S. Chen, J. Roberts, X. Kong, and D. Greenblatt. Regulation of classic and alternative bile acid synthesis in hypercholesterolemic rabbits: effects of cholesterol feeding and bile acid depletion.

Sitosterolemia (phytosterolemia) is a rare autosomal recessively inherited disorder that is characterized by premature coronary artery disease, xanthomas, and increased plasma plant sterols and 5α-stanols. Affected individuals show an increased absorption of both cholesterol and sitosterol from the diet, decreased bile clearance of these sterols and their metabolites resulting in markedly expanded whole body cholesterol and sitosterol pools. Biochemical studies have shown that the regulation of the cholesterol biosynthetic pathway may be abnor mal in this condition. In particular, the activities and mRNA for the biosynthetic enzymes, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and HMG-CoA synthase are low in liver biopsy specimens isolated from affected individuals, suggesting replete intracellular cholesterol pools. However, the membrane expression of hepatocyte low density lipoprotein receptors was increased, suggesting discordant regulation. Segregation analyses in three families for the genes for HMG-CoA reductase, HMG-CoA synthase, and LDL-receptor excluded these as sites of mutation. In view of the previously described discordant regulation of the above genes in sitosterolemia, the two major regulatory genes for this pathway, sterol regulatory element binding proteins (SREBP-1 and -2), were also examined. These genes did not segregate with the disease and were thus excluded. Two other genes involved in cholesterol absorption and chylomicron secretion, namely acyl coenzyme A:cholesterol acyltransferase (ACAT) and microsomal triglyceride transfer protein (MTP) were also examined for segregation and similarly excluded. Although the gene defect in sitosterolemia therefore remains to be elucidated, important candidate genes have been excluded.—Patel, S. B., A. Honda, and G. Salen. Sitosterolemia: exclusion of genes involved in reduced cholesterol biosynthesis. J. Lipid Res. 1998. 39: 1055–1061.

Sitosterolemia is a recessively inherited disorder characterized by abnormally increased plasma and tissue plant sterol concentrations. Patients have markedly reduced whole body cholesterol biosynthesis associated with suppressed hepatic, ileal, and mononuclear leukocyte 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-controlling enzyme in cholesterol biosynthetic pathway, coupled with significantly increased low density lipoprotein (LDL) receptor expression. To investigate the mechanism of down-regulated cholesterol biosynthesis, we assayed several other key enzymes in the cholesterol biosynthetic pathway including acetoacetyl-CoA thiolase, HMG-CoA synthase, squalene synthase, and 7-dehydrocholesterol Δ7-reductase activities in liver and freshly isolated mononuclear leukocytes from four sitosterolemic patients and 19 controls. Hepatic acetoacetyl-CoA thiolase, HMG-CoA synthase, reductase, and squalene synthase activities were significantly decreased (P < 0.05) –39%, –54%, –76%, and –57%, respectively, and 7-dehydrocholesterol Δ7-reductase activity tended to be lower (–35%) in the sitosterolemic compared with control subjects. The reduced HMG-CoA synthase, reductase, and squalene synthase activities were also found in mononuclear leukocytes from a sitosterolemic patient. Thus, reduced cholesterol synthesis is caused not only by decreased HMG-CoA reductase but also by the coordinate down-regulation of entire pathway of cholesterol biosynthesis. These results suggest that inadequate cholesterol production in sitosterolemia is due to abnormal down-regulation of early, intermediate, and late enzymes in the cholesterol biosynthetic pathway rather than a single inherited defect in the HMG-CoA reductase gene.—Honda, A., G. Salen, L. B. Nguyen, G. S. Tint, A. K. Batta, and S. Shefer. Down-regulation of cholesterol biosynthesis in sitosterolemia: diminished activities of acetoacetyl-CoA thiolase, 3-hydroxy-3-methylglutaryl-CoA synthase, reductase, squalene synthase, and 7-dehydrocholesterol Δ7-reductase in liver and mononuclear leukocytes. J. Lipid Res. 1998. 39: 44–50.

The metabolism of Δ7-cholestenol, cholesterol, and cholestanol was examined in a patient with cerebrotendinous xanthomatosis after intravenous pulse-labeling with a mixture of dl-[2-14C]mevalonate and stereospecific 3S,4S,3R,4R-[4-3H]-mevalonate. Silver nitrate and reversed-phase thin-layer chromatography were used to purify the sterols isolated from the feces, and their identities were confirmed by gas–liquid chromatography–mass spectrometry. The specific activities were determined and plotted as a function of time. Isotope ratio measurements and specific activity decay curves showed that sterol synthesis proceeded in the following sequence: mevalonate, squalene, lanosterol, Δ7-cholestenol, cholesterol, cholestanol. Labeled cholesterol precursors might be advantageously used to measure changes in cholesterol synthesis because they appear to equilibrate rapidly and have very short turnover times.

The influence of clofibrate on cholesterol metabolism in patients with hyperlipidemia was studied by means of sterol balance and isotope kinetic techniques and by measurements of flow rates of cholesterol through the biliary tract. Long-term balance studies were carried out on a metabolic ward in 24 patients with all currently recognized types of hyperlipidemia; in five other patients with hypercholesterolemia, pool sizes and turnover rates of cholesterol were defined by compartmental analysis before and after three years' daily administration of the drug.Except in fat-induced hypertriglyceridemia (two patients), clofibrate caused reduced plasma levels of triglycerides and cholesterol in all categories of hyperlipidemia. As a general rule, excretion of cholesterol into bile and feces was significantly increased and fecal bile acid excretion was decreased, regardless of the type of lipoprotein abnormality. Despite a net increase in steroid excretion in most patients with hyperlipidemia, cholesterol synthesis was not increased; indeed, in many patients synthesis appeared to be decreased. While the data obtained in 29 patients were not always consistent, the bulk of the evidence suggests that, in all forms of hyperlipidemia except fat-induced hyperglyceridemia, the drug causes an increased output of cholesterol while simultaneously inhibiting any compensatory increase in cholesterol synthesis. Therefore, it appeared that the increased excretion of steroids was most likely derived from cholesterol stored in tissues. This conclusion was strengthened by finding that long-term administration of the drug can cause marked reduction in body pools of cholesterol.These findings are reflected clinically by resolution of skin and tendon xanthomatosis. However, it is not yet known whether the accumulation of cholesterol in arterial walls that is part of the process of atherogenesis can be inhibited or reversed by the drug.